UNIT II Chromatography Principle, theory, instrumentation ...
Transcript of UNIT II Chromatography Principle, theory, instrumentation ...
UNIT II
Chromatography – Principle, theory, instrumentation and applications in chemical
analysis of the following – column, paper, thin layer and ion-exchange – GC, GLC and
HPLC. Purification of common organic solvents.
Atomic absorption spectroscopy and Flame emission spectroscopy – basic
principle – Instrumentation and applications. Comparison between AAS and FES.
REFERENCES
1. Willard, Merrit and Dean, Instrumental Methods of Chemical Analysis
2. Chatwal, Instrumental Methods of Analysis
3. Sharma, Instrumental Methods of Chemical Analysis
4. Kenner, Analytical Separations and Determinations
5. Sharma, Chromatography
PRINCIPLES AND APPLICATION
OF CHROMATOGRAPHY
CHROMATOGRAPHY
Laboratory technique for the Separation of mixtures
Chroma -"color" and graphein - "to write”.
Colour bands - separation of individual compounds
Measured or analysed.
PURPOSE OF CHROMATOGRAPHY
• Analytical
Determine Chemical composition of a sample
• Preparative
Used to purify sufficient quantities of a substance
TSWETT EXPERIMENT
• Chromatograph - equipment that enables a sophisticated
separation
EX. Gas chromatography or Liquid chromatography
• Eluent - Fluid entering column/ solvent that carries the analyte.
• Eluate - Mobile phase leaving the column.
• Stationary phase - Immobilized phase
Immobilized on the support particles or on the inner wall of the
column tubing.
Examples : Silica layer - Thin Layer Chromatography
CHROMATOGRAPHY TERMS
• Mobile phase
Moves in a definite direction. Liquid (LC), Gas (GC).
• The mobile phase moves through the chromatography
column (the stationary phase) where the sample interacts
with the stationary phase and is separated.
• Retention time : Time takes for a particular analyte to
pass through the system (from the column inlet to the
detector) under set conditions.
• Sample (Anylate) :Substance analyzed in
chromatography.
• Solvent : Any substance capable of solubilizing another
substance.
Chromatogram
Visual output of the chromatograph.
Separation - Different peaks or patterns on the
chromatogram correspond to different components of the
separated mixture.
Chromatogram - Detector signal vs. retention time or volume
time or volume
Det
ecto
r Si
gnal 1 2
X- axis - Retention time
Y-axis - Signal
Signal is proportional to the concentration of the specific analyte
separated.
HOW TO DESCRIBE A CHROMATOGRAM
PRICNIPLES OF CHROMATOGRAPGHY
• Physical method of separation that distributes components
to separate between two phases moves in a definite
direction.
• Substances are separated based on their differential
distribution between two phases
• Substances will move with the mobile phase at different
rate depending upon their Partition or Distribution co-
efficients.
PRINCIPLES
• The samples are subjected to flow by mobile liquid phase onto
or through the stable stationary phase.
• Separation of fractions of mixture based on their relative affinity
towards the two phases during their travel.
• The fraction with greater affinity to stationary phase travels
slower and shorter while that with less affinity travels faster and
longer. The separation is based on Differential partitioning
between the mobile and stationary phases.
FACTORES AFFECTING THE SEPARATION
• Intermolecular interaction between the two phases
• Extent of dispersion of solute molecules over the
stationary phase
CLASSIFICATION OF CHROMATOGRAPHY
• Techniques by Chromatographic bed shape
– Column chromatography
– Planar chromatography
• Paper chromatography
• Thin layer chromatography
• Techniques by Physical state of mobile phase
– Gas chromatography
– Liquid chromatography
• Affinity chromatography
– Supercritical fluid chromatography
TECHNIQUES BY CHROMATOGRAPHIC BED SHAPE
A.COLUMN CHROMATOGRAPHY
PRINCIPLES
Solid materials (Adsorbants) – Ability to hold the molecules
at their surface
Attractive forces (Vanderwalls & Hydrogen )
Functional groups (Hydroxyl/ Aromatic)
Silica
• Stationary bed is within a tube.
• Solvent is driven through the column by applying Positive
pressure.
• Separations - 20 minutes
• Modern flash chromatography :
Pre-packed plastic cartridges,
Solvent is pumped through the cartridge.
Quicker separations
Less solvent usage.
• Column :
o Diameter - 5 mm to 50 mm
o Height - 5 cm to 1 m with a tap
o Filter (a glass frit or glass wool plug)
• The individual components are retained by the stationary
phase differently and separate from each other while they are
running at different speeds through the column with the eluent.
• During the entire chromatography process the eluent is
collected in a series of fractions. The composition of the eluent
flow can be monitored and each fraction is analyzed for
dissolved compounds, e.g., UV absorption, or fluorescence.
STATIONARY PHASE
Silica gel, Alumina. Cellulose
SOLVENTS
• Hydroxyl groups - Alcohol
• Carboxyl group - Acetone
• Non polar Compounds – Hexane
Heptane
Toulene
• Flow rate - Separation.
• Pump or compressed gas (e.g. Air, Nitrogen, Argon)
• A faster flow rate of the eluent:
Minimizes the time required to run a column
Minimizes diffusion
Better separation.
Retention Time: The time from the start of signal detection by the
detector to the peak height of the elution concentration profile of
each different sample.
Curve Width: The width of the concentration profile curve of the
different samples in the chromatogram in units of time.
RESOLUTION (RS) :
Rs = 2(tRB – tRA)/(wB + wA)
Where:
tRB = Retention time of solute B
tRA = Retention time of solute A
wB = Gaussian curve width of solute B
wA = Gaussian curve width of solute A
Plate Number (N):
N = (tR)2/(w/4)2
Plate Height (H):
H = L/N
Where L is the length of the column.
B. PLANAR CHROMATOGRAPHY
• Separation technique - Stationary phase is present as or on a
plane.
• Paper – Paper Chromatography
• Layer of solid particles spread on a support such as a glass
plate - Thin layer Chromatography.
• Different compounds in the sample mixture travel different
distances according to how strongly they interact with the
stationary phase as compared to the mobile phase.
• Retention factor (Rf)
• This paper is made of cellulose, a polar substance, and the
compounds within the mixture travel farther if they are non-
polar.
• More polar substances bond with the cellulose paper more
quickly, and therefore do not travel as far.
PRINCIPLE
• Retention factor :
• Rƒ = Distance travelled by a Solute
Distance travelled by a Solvent
• Rƒ = zero, - Solute remains in the stationary phase and
thus it is immobile.
• Rƒ = 1 - Solute has no affinity for the stationary phase
and travels with the solvent front.
• b) THIN LAYER CHROMATOGRAPHY
• Widely employed laboratory technique
• Stationary phase - Adsorbent - Silica gel
Alumina
Cellulose
• Widely used in pharmaceutical & food stuff industry
Advantages :
Simple, Rapid and Cheap
Faster runs
Better separations
Choice between different adsorbents.
Better resolution
Allow for quantification
Used to identify the unknown compounds and to determine
the purity of mixture.
TLC Plate - Aluminium or glass - coated by stationary phase.
Coated material : 0.1-0.3mm in thickness
Fluorescent indicator that will make it florescence during the UV
light exposure.
MOBILE PHASE
• Volatile Organic solvents
STATIONARY PHASE
Silica gel, Alumina, or Cellulose on a flat, inert substrate.
SPRAYS
• RETENTION FACTOR :
• Rƒ = Distance travelled by a Solute
• Distance travelled by a Solvent
• Rƒ = zero, Solute remains in the stationary phase and
• thus it is immobile.
• Rƒ = 1 Solute has no affinity for the stationary phase
and travels with the solvent front.
• 2.TECHNIQUES BY PHYSICAL STATE OF MOBILE
PHASE
A. GAS CHROMATOGRAPHY
• Gas-Liquid chromatography, (GLC)
• Mobile phase – Gas (Helium) Carrier Gas Pressure = 4 kg/cm2
• Stationary phase - Column, which is typically "packed" or "capillary".
• The stationary phase is adhered to the inside of a small-diameter glass
tube (a capillary column) or a solid matrix inside a larger metal tube (a
packed column).
• Partition Coefficient of Volatile analyte between a solid stationary
phase (Silicone) and a mobile gas (Helium).
• Advantages• High sensitivity,
• High Resolution,
• High speed
• High Accurasy,
• Highly Quantitative
APPARATUS
• Gas Chromatograph, GC analyzer, Normal syringes and one micro syringe,Beakers, Sample bottles and Electronic weight.
CHEMICALS
• Methanol, Isopropyl Alcohol and water
SAMPLE:
• Gases, Liquid, Solids
• M.Wt: 2-800
• Volatile
APPLICATION
• Quantitative & Qualitative analysis of low polarity compounds
• Analytical chemistry, Biochemistry, Petrochemical,
Environmental monitoring
• Measure picomoles of a substance in a 1 ml liquid sample, or
parts-per-billion concentrations in gaseous samples
• Measuring toxic substances in soil, air or water.
APPLICATION OF GC- MS
Environmental monitoring : Oraganic Pollutants
Criminal forensics : Analyze the particles (Fibre) from a human
body in order to help link a criminal to a crime.
Law enforcement : Detection of illegal narcotics,
Forensic toxicology : Find drugs and/or poisons in biological
specimens of suspects, victims, or the deceased.
Sports anti-doping analysis : Test athletes' urine samples
Security : Explosive detection (September 11 development) systems
have become a part of all US airports.
Food, beverage and perfume : from spoilage or Adultration -
aromatic compounds, esters, fatty acids, alcohols, aldehydes, terpenes
Medicine : Congenital metabolic diseases
In Born error of metabolism
• B. LIQUID CHROMATOGRAPHY
• Mobile phase - Liquid.
• Column or a plane.
• Very small packing particles and a relatively high pressure -
High Performance Liquid Chromatography (HPLC).
LC- MS
Mass spectra is obtained rapidly
Small amount of material is required to form the spectra.
Data collected is highly informative with respect to
molecular structure.
APPLICATION
• Pharmacokinetics : How quickly a drug will be cleared from the
hepatic blood flow and organs of the body.
• Proteomics : Peptide mass fingerprinting
• Drug development: Peptide Mapping, Glycoprotein Mapping,
Natural Products Dereplication, Bioaffinity Screening, In Vivo
Drug Screening, Metabolic Stability Screening, Metabolite
Identification, Impurity Identification, Degradant Identification,
Quantitative Bioanalysis, and Quality Control.
• Fungal toxins
• Pesticides, Herbicides
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
HPLC V/S LC TECHNIQUE
Columns : Small diameter (4.6 mm), stainless steel, glass or
titanium.
Column packing with very small (3, 5 and 10 μm) particles
Relatively high inlet pressures and controlled flow of the
mobile phase.
Detecting very small amounts
High resolution
Rapid analysis
Speed, efficiency, sensitivity and ease of operation
High degree of versatility
Easily separate a wide variety of chemical mixtures
400 atmospheres.
PUMP PRESSURE
"Ultra High Performance Liquid Chromatography" systems1000 atmospheres.
• ELUTION : Isocratic and Gradient.
ISOCRATIC :
• ISO ==> SAME
• - Solvent Composition Stays the Same for the Entire Run
EX: 60:40 Alcohol:Water
GRADIENT :
• Solvent Composition Changes Throughout the Run
TYPES OF HPLC
Nature of the stationary phase
Separation process
Adsorption chromatography
Ion-exchange chromatography
Size exclusion chromatography
APPLICATION
Protein separation
Insulin purification
Plasma fractionation
Enzyme purification
SIZE EXCLUSION CHROMATOGRAPHY
• Gel filtration or gel permeation chromatography
• Separation - Molecular size of its components.
• Larger molecules are rapidly washed through the column, smaller
molecules penetrate inside the porous of the packing particles and
elute later.
APPLICATIONS
• AFFINITY CHROMATOGRAPHY
• Based on specific & non-covalent binding of the proteins to
other molecules – Ligands ( His-tags, biotin or antigens)
• Physical properties of the analyte.
• Biochemistry in the purification of proteins (Enzymes)
bound to tags.
• After purification, some of these tags are usually removed
and the pure protein is obtained.
SUPERCRITICAL FLUID CHROMATOGRAPHY
Used for the analysis and purification of low to moderate molecular weight ,
thermally labile molecules.
Principles are similar to those of (HPLC)
Mobile phase - High pressure liquid or Super critical Carbon Dioxide.
Modifiers – Methanol, Ehanol, isopropyl alcohol, acetonitrile and
Chloroform.
APPLICATION
Use in industry primarily for separation of Chiral (Asymmetric Carbon atoms) molecules.
• Serine
• Soman
• Glyceraldehyde
• Phosphours (Phosphine)
• Sulfar metal
• Cobalt
• Enkephalins
DETECTOR
• Gas Chromatography or liquid Chromatography
• To visualize components of the mixture being eluted off the
chromatography column.
DETECTORS
• Flame ionization detector
• Aerosol-based detector
• Flame photometric detector ( FPD).
• Atomic-emission detector (AED).
• Mass spectrometer ( MS) detector
• Nitrogen Phosphorus Detector,
• Evaporative Light Scattering Detector (ELD) : LC.
DETECTORS
• UV detectors
• Thermal conductivity Detector, (TCD)
• Fluorescence detector
• Electron Capture Detector, (ECD)
• Photoionization Detector, (PID)
• Refractive index Detector (RI or RID)
• Radio flow Detector
• Chiral Detector
ATOMIC ABSORPTION SPECTROSCOPY
INTRODUCTON: • Atomic absorption spectroscopy is deals with the
absorption of specific wave length of of radiation by neutral atoms in the ground state. This phenomenon is similar to UV spectroscopy, where absorption of radiation by molecules occur.
• Neutral atoms are obtained by spraying the sample solution of element using a burner. Specific wavelength of radiation is generated by using a hollow cathode lamp. for determination of every element , separate hollow cathode lamp is required.
PRINCIPLE: • When solution of metalic salt is sprayed on to a flame,
fine droplets are formed , due to the thermal energy of the flame , the solvent in the flame is evaporated , leaving a fine residue, which are converted to neutral atoms.
• These neutral atoms absorb radiation of specific wavelength , emitted by hollow cathode lamp(HCL).hollow cathode lamp is filled with the vapour of element , which gives specific wavelength of radiation.
• For the determination of every element, hollow cathode lamp is selected, which contains vapour of the element to be analysed although this appear to be demerits of AAS , specificities can be achieved only by the use of HCL.
• The intensity of light absorbed by the neutral atom is directly proportion to the concentration of the element and obeys Beer's law over a wide concentration range.
• The intensity of radiation absorbed by neutral atoms is measured using photometric detectors (PMT)
• In AAS the temperature of the flame is not critical , since the thermal energy of flame isused to atomise the sample solution to fine droplets , to form a fine residue and later to neutral atoms.
• The exitation of neutral atoms is brought about only by radiation from hollow cathode lamp and not by the thermal energy of the flame.
INSTRUMENTATION
HOLLOW CATHODE LAMP
•The lamp or source of light in AAS is a hollow cathode lamp. • The cathode is made up of specific element or alloys of elements or coating of element on cathode. • When current of 500 V is applied between anode and cathode, metal atoms emerge from hollow cup and collides with filler gas which is argon or neon • Due to these collisions, numbers of metal atoms are exited and emitt their characteristic radiation .
• These characteristic radiation is absorbed by neutral atoms of the same element in ground state , which occur in the flame, when sample solution is sprayed.
• It is not possible to use a source of light with a monochromator because this arrangement gives a radiation with a band width of 1nm, where as the hollow cathode lamp gives a band width of 0.001 to 0.01nm, which is highly desirable to achieve specificity.
• Moreover, light source should provide a line width less than the absorption line width of the element to be determined
BURNER (WITH FUEL AND OXIDANT):
• There are different burners are available, which are used to spray the sample solution into fine droplets, mix with fuel and oxidant , so that a homogeneous flame of stable intensity is obtained.
• The most common burners are
1. TOTAL CONSUMPTION BURNER
2. LAMINAR FLOW BURNER
• If the temperature of the flame is too low, it may not cause exitation of neutral atoms. If temperature is too high , it may cause ionisation of atoms and thus sufficient atoms in exited state may not occur.
• This makes it necessary to select ideal combination of oxidant and fuel which gives the desired temperature.
CHOPPER:
• The chopper in the instrument is rotate like a fan , allows alternatively radiation from flame alone or the radiation from HCL and the flame.
• This produces a pulsating current or signal, which is used to measure the intensity of light absorbed by elements, without interference by radiation from the flame itself.
MONOCHROMATOR
• Some elements have single absorption line , but several elements have more than one absorption line .
• Hence it is necessary to select the spectral line for absorption have measurement.
• Moreover it is necessary to isolate the line spectrum of element from that of the emission by the gas in the lamp , or from the background signal of the flame.
• Hence a monochromator which can provide good resolution of 1nm or less is required
DETECTOR & READOUT DEVICE
• The intensity of radiation absorbed by elements, in UV or visible region (190-780nm) can be detected using photometric detectors.
• The readout device is capable of displaying the absorption spectrum as well as a specified wavelength.
INTERFERENCE
• Spectral interference
• Chemical interference
• Ionic interference
• Matrix interference
• Solvent interference
• Dissociation of metal compound
APPLICATION OF AAS
• Estimation of trace elements in biological fluid like blood, urine, etc.
• Estimation of trace elements like Copper , Nickle and Zinc in food products.
• Estimation of Magnesium , Zinc in blood.
• Estimation of Zinc in Zinc insulin solution.
• Estimation of Mercury in Thiomersal solution.
• Estimation of Lead in Calcium carbonate and petrol.
• Estimation of elements in soil samples , water supply , effluents , ceramics , etc.